Antibody-Membrane Switch (AMS) technology for optimized antibody engineering Abstract Generation of high-productivity cell lines remains a major bottleneck in therapeutic antibody development. Conventional cell line development depends on gene amplification methodologies using dihydrofolate reductase (DHFR) or glutamine synthetase (GS). Higher productivity is associated with an increased gene copy number. However, lack of selection pressure under the conditions of large scale manufacturing leads to clonal instability. We have developed a novel method for cell line development, Antibody Membrane Switch (AMS) technology that does not rely on gene amplification. This FACS-based, high-throughput method is facilitated by cell surface antibody expression to rapidly and efficiently isolate high producing cells. The switch between membrane expression and secretion is achieved by alternative splicing and specific DNA recombination. The antibody of interest is initially displayed on the cell surface to facilitate FACS. Isolated high producing cells are then seamlessly transformed into production cells after removing the membrane-anchoring domain sequence via a DNA recombinase. AMS technology has been applied in a number of antibody cell line development projects which typically last 2-3 months. The top manufacturing cell lines exhibit very high specific productivity of 40-60 pg./cell/day resulting in production titers of 2-4 g/L in 10-day batch culture. In Phase I studies the underlying AMS methods were reduce to practice. Furthermore, we have optimized the AMS process using two biosimilar antibodies. In Phase II, these optimized methods will be applied to screening of CHO surface displayed antibody libraries. An affinity maturation library and a nave human antibody library will be constructed and utilized to screen by FACS for therapeutic antibody candidates. The screening will be carried out with soluble antigen for high affinity binding or antigen (e.g., GPCR) presenting cells for desired bio-activities. The isolated positive cells will be transformed into antibody production cells by DNA recombinase to remove the membrane anchorage. This successful application of AMS technology will permit the discovery of antibodies to be seamlessly linked to downstream cell line development providing the basis for rapid, facile cell line production directly from a novel antibody-discovery platform. Phase I: 1R43AI109982-01A1